Stem cells have the ability to divide for indefinite periods in culture and to give rise to specialized cells. Typically, stem cells are divided into two main groups: adult stem cells and embryonic stem cells. Stem cells may also be generated through artificial means such as nuclear transfer, cytoplasmic transfer, cell fusion, parthenogenesis and reprogramming. Isolated stem cells can give rise to many types of differentiated cells, and can be used to treat many types of diseases.
Adult stem cells are undifferentiated but are present in differentiated tissues, and are capable of differentiation into the cell types from the tissue that the adult stem cell originated. Adult stem cells have been derived from various sources, such as the nervous system (McKay, 1997, Science 276:66-71; Shihabuddin, et al., 1999, Mol. Med Today 5:474-480); bone marrow (Pittenger, et al., 1999, Science 284:143-147; Pittenger, M. F. and Marshak, D. R. (2001) In: Mesenchymal stem cells of human adult bone marrow. Marshak, D. R., Gardner, D. K., and Gottlieb, D. eds. (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press) 349-374); adipose tissue (Gronthos, et al., 2001, J Cell. Physiol. 189:54-63), dermis (Toma, et al., 2001, Nature Cell Biol. 3:778-784); pancreas and liver (Deutsch, el al., 2001, Development 128:871-881). Stem cells have also been isolated from umbilical cord (Rogers, et al., 2004, Best Pract Res Clin Obstet Gynaecol. 18(6):893-908; Wang et al., 2004, Stem Cells 22(7):1330-1337; Surbek, et al, 2002, Ther Umsch. 59(11):577-582; and placenta (Yen et al., 2005, Stem Cells 23(1):3-9), each of which is incorporated by reference herein in its entirety. It is believed that stem cells of the adult type are also found in smooth muscle tissue, striated muscle tissue, cardiac muscle tissue, bone tissue, bone spongy tissue, cartilage tissue, pancreatic ductal tissue, spleen tissue, thymus tissue, tonsil tissue, Peyer's patch tissue, lymph nodes tissue, thyroid tissue, epidermis tissue, dermis tissue, subcutaneous tissue, heart tissue, lung tissue, vascular tissue, endothelial tissue, blood cells, bladder tissue, kidney tissue, digestive tract tissue, esophagus tissue, stomach tissue, small intestine tissue, large intestine tissue, adipose tissue, uterus tissue, eye tissue, lung tissue, testicular tissue, ovarian tissue, prostate tissue, connective tissue, endocrine tissue, and mesentery tissue.
Several patents disclose various aspects of adult stem cells. For example, U.S. Pat. No. 5,556,783 discloses methods of culturing hair follicle stem cells, while U.S. Pat. No. 5,486,359 discloses methods of isolating human mesenchymal stem cells. U.S. Pat. Nos. 4,714,680, 5,061,620, and 5,087,570 provide examples of hematopoietic stem cells. Each of these patents is incorporated by reference herein in its entirety.
Embryonic stem cells are undifferentiated cells derived from the embryo. Typically these cells are extracted from the inner cell mass of a blastocyte and when cultured under the unique conditions, either alone or in combination with a variety of feeder cells, the embryonic stem cells maintain euploid karyotype, do not undergo senescence, and retain the ability to differentiated into cells of the endodermal, ectodermal, and mesodermal lineages.
These cells have the potential to become a wide variety of specialized cell and tissue types, which can then be used for basic research, drug discovery, and treatment (or prevention) of many types of diseases. Patents describing aspects of embryonic stem cells include U.S. Pat. No. 6,506,574 to Rambhatla, U.S. Pat. No. 6,200,806 to Thomson, U.S. Pat. No. 6,432,711 to Dinsmore, and U.S. Pat. No. 5,670,372 to Hogan, each of which is incorporated by reference herein in its entirety. Importantly, murine embryonic stem cells can be cultured indefinitely under the presence of leukemia inhibitory factor (LIF), which maintains their undifferentiated state. In contrast, human embryonic stem cells are not responsive in the same manner to LIF, thus stimulating the invention of numerous methodologies to expand them. Unfortunately, many such methodologies involve the use of either murine feeder cells or other animal components, hence limiting the therapeutic potential of these cells. Furthermore, even when established cell lines, such as the federally approved embryonic stem cells, are cultured in murine-free conditions, contamination is still present as recently reported (Martin, et al., 2005, Nat Med 11:228-232, which is incorporated by reference herein in its entirety). Accordingly, one object of the invention disclosed is to provide novel methods of expanding stem cells in absence of animal components, said invention being applicable to a variety of stem cells, including embryonic stem cells.
The importance of technologies associated with expansion of stem cells, both of adult and/or embryonic derivation is illustrated by the numerous preclinical and clinical uses of these cells in treatment of a wide range of diseases.
One of the earliest clinical uses of stem cells was for performing bone marrow transplants in patients with hematological malignancies in which hematopoietic stem cells derived from the donor bone marrow were administered into the recipient subsequent to providing said recipient with a sufficient dose of radiation and/or chemotherapy in order to ablate not only the hematological malignancy but also non-malignant hematopoiesis. The administration of, non-malignant hematopoietic stem cells resulted in donor-specific hematopoiesis and in some patients, cure of the malignancy. This was first described by Thomas et al in 1957, who reported that large volumes of donor bone marrow could be safely infused in patients with acute leukemia following myeloablation and that donor-specific hematopoiesis was established (Thomas, et al., 1957, N Engl J Med 257:491-496, which is incorporated by reference herein in its entirety). The identification of similar hematopoietic stem cell activity in the peripheral blood led to development of techniques used to mobilize and harvest peripheral blood hematopoietic stem cells for use in transplantation settings. For example, the use of GM-CSF and G-CSF in enhancing the number of peripheral blood hematopoietic stem cells was reported in the clinical situation of autologous transplantation subsequent to high dose chemotherapy (Peters, et al., 1993, Blood 81:1709-1719; Sheridan, et al., 1992, Lancet 339:640-644, each of which is incorporated by reference herein in its entirety).
In addition to treatment of hematological malignancies, stem cells have been utilized in the context of therapy for solid tumors. The dose limiting variable in cancer chemotherapy is bone marrow toxicity. Accordingly, in 1958, Kurnick et al performed an autologous bone marrow transplant to demonstrate ability of infused bone marrow to allow use of very high doses of chemotherapy and/or radiation therapy (Kurnick, et al., 1958, Ann Intern Med 49:973-986, which is incorporated by reference herein in its entirety). The use of autologous hematopoietic cell transplants combined with high dose chemo/radiotherapy for solid tumors has been extensively investigated for breast (Peppercorn, et al., 2005, Cancer 104:1580-1589; Dillman, et al., 2005, Am J Clin Oncol 28:281-288), colon (Leff, et al., 1986, J Clin Oncol 4:1586-1591; Franchi, et al., 1994, Eur J Cancer 30A:1420-1423), lung (Ziske, et al., 2002, Anticancer Res 22:3723-3726), nasopharyngeal cancer (Chen, et al., 2003, Jpn J Clin Oncol 33:331-335), and other types of cancers (Gratwohl, et al., 2004, Ann Oncol 15:653-660), each of which is incorporated by reference herein in its entirety.
The identification of the type 1 transmembrane protein/adhesion molecule, the sialomucin CD34 as a marker of hematopoietic stem cells led to the use of CD34+ cell selection as a means of concentrating hematopoietic stem cell activity (Civin, et al., 1984, J Immunol 133:157-165, which is incorporated by reference herein in its entirety). Specifically, it was demonstrated that although bone marrow mononuclear cells contain approximately 1-4% CD34+ cells, the administration of these cells, but not bone marrow depleted of CD34+ cells, into lethally irradiated baboons led to hematopoietic reconstitution (Berenson, et al., 1988, J Clin Invest 81:951-955, which is incorporated by reference herein in its entirety). Clinical development of purified CD34+ cells as a source of stem cells was originally sought as a method of performing bone marrow transplant without contamination of donor T cells. This would in theory stop development of graft versus host disease, one of the main causes of allogeneic transplant associated morbidity and mortality (Ferrara, et al., 2005, Clin Adv Hematol Oncol 3:415-419, 428, which is incorporated by reference herein in its entirety). Unfortunately, clinical evidence demonstrated that patients receiving purified CD34+ stem cell grafts, although having a lower incidence of graft versus host disease, also had a higher incidence in leukemic relapse due to an immunologically mediated graft versus leukemia effect that is absent when donor bone marrow grafts are depleted of T cells (Martino, et al., 2000, Haematologica 85:1165-1171; Butt, et al., 2003, Leuk Lymphoma 44:1509-1513, each of which is incorporated by reference herein in its entirety). The critical importance of bone marrow derived T cells in the induction and upkeep of graft versus leukemia effects was illustrated in studies of leukemic patients who have relapsed and were subsequently treated by infusion of donor T cells. This induced a long-term remission in the patients that had major relapse (Kolb, H. J., 1998, Vox Sang 74 Suppl 2:321-329; Guglielmi, et al., 2002, Blood 100:397-405, each of which is incorporated by reference herein in its entirety). Furthermore, it was also observed that under some conditions, bone marrow derived non-CD34 cells of the osteoblast lineage have a role in facilitating engraftment in allogeneic settings (Good, R. A., 2000, World J Surg 24:797-810, which is incorporated by reference herein in its entirety). Despite these potential drawbacks, clinical use of CD34+ cells both from mobilized peripheral blood, as well as bone marrow, during autologous transplantation for high dose chemo/radiation therapy was considered to be a useful approach (Korbling, et al., 2001, Blood 98:2900-2908; Pecora, et al., 2001, Bone Marrow Transplant 27:1245-1253; Pecora, A. L., 1999, Bone Marrow Transplant 23 Suppl 2:S7-12, each of which is incorporated by reference herein in its entirety). This is due to the fact that in this setting, neither facilitator cells are needed, since the graft is autologous, and the CD34+ selection substantially clears the marrow of contaminating tumor cells, so that the risk of tumor relapse is lessened as opposed to using non-purified bone marrow (Preti, et al., 2001, Cytotherapy 3:85-95; Siena, et al., 2000, J Clin Oncol 18:1360-1377; Vannucchi, et al., 1998, Br J Haematol 103:610-617, each of which is incorporated by reference herein in its entirety).
The use of hematopoietic stem cells has also been described for “reprogramming” the immune system to induce an antigen-specific state of non-responsiveness called tolerance. Specifically, this use can be divided into two main areas: the use of stem cells to induce donor-specific tolerance during allogeneic or xenogeneic transplantation, and the use of stem cells to induce tolerance in situations of autoimmunity. Although common mechanisms of tolerance maintenance such as generation of T regulatory cells, effector T cell depletion, and effector T cell anergy have been described in both types of tolerance, the mechanism of induction seems to be different; therefore we will describe them individually.
The possibility of bone marrow hematopoietic stem cells having the utility of inducing tolerance to a grafted organ was first elaborated on by Owens in the 1940s. In studies demonstrating that in utero mixing of blood in the context of shared circulation between two genetically different cows, he observed bilateral transplantation tolerance in adulthood. Accordingly, he postulated that the original sharing of circulation may have contributed to the state of tolerance which theoretically should not have existed due to the genetic disparity between the siblings. Furthermore, definitive roles for using stem cells to induce tolerance came from Billingham and Medawar in the 1950s in experiments showing injection of donor bone marrow cells into neonates allowed for tolerance to the donor antigen when the animal reached adulthood (Slavin, S., 2002, Int J Hematol 76 Suppl 1:172-175, which is incorporated by reference herein in its entirety). In animal models it has been demonstrated that bone marrow cells contribute to generation of a donor-specific tolerogenic state which is associated with chimeric hematopoiesis. The combination of donor-specific bone marrow transplant, with solid organs, has been used in some clinical situations to induce complete tolerance to the grafted organ without the need for chronic, continuous immune suppression (George, et al., 2002, Immunol Res 26:119-129, which is incorporated by reference herein in its entirety). Unfortunately, wide spread use of bone marrow induced tolerance is limited by the fact that bone marrow transplantation is associated with a high degree of morbidity and mortality during the myeloablative phase. In addition, the possibility of graft versus host disease is another pitfall to the full-scale implementation. In order to overcome this, several methods of inducing partial chimerism, or mini-chimerism are being investigated through the use of non-myeloid ablative techniques such as donor-specific transfusions combined with anti CD154 antibodies (Seung, et al., 2003, J Clin Invest 112:795-808, which is incorporated by reference herein in its entirety). Induction of organ tolerance by hematopoietic stem cells is believed to occur through both thymic dependent (Noris, et al., 2001, J Am Soc Nephrol 12:2815-2826, which is incorporated by reference herein in its entirety), and independent (van Pel, et al., 2003, Transpl Immunol 11:375-384, which is incorporated by reference herein in its entirety) mechanisms. Specifically, donor hematopoietic cells generate a variety of both lymphoid and non-lymphoid cells that express the same antigens found in the donor organ, but somehow redirect the immune system not to attack these specific antigens, while maintaining responses against other antigens not related to the graft. One mechanism that is postulated to occur is the thymic stromal tissue in the recipient becomes populated with donor-derived cells. These cells then act at the level of negative selection in order to induce apoptosis of T cells reactive to the donor antigen in a similar way to which the immune system deletes autoreactive T cells during thymic selection (Shizuru, et al., 2000, Proc Natl Acad Sci USA 97:9555-9560, which is incorporated by reference herein in its entirety). Another mechanism of tolerance involves the persistent presentation of donor antigen in absence of costimulatory molecules. This was demonstrated in one situation by the fact that persistence of T cells from the donor bone marrow is essential in maintaining tolerance (Xu, et al., 2004, J Immunol 172:1463-1471, which is incorporated by reference herein in its entirety). The mesenchymal component of the bone marrow produces a cell population that consitutively secretes immune inhibitory factors such as IL-10 and TGF-b while presenting antigens (Liu, et al., 2004, Transplant Proc 36:3272-3275; Togel, et al., 2005, Am J Physiol Renal Physiol 289:F31-42, each of which is incorporated by reference herein in its entirety). This is believed to further inhibit immunity in an antigen specific manner. During T cell activation, two general signals are required for the T cell in order to mount a productive immune response, the first signal is recognition of antigen, and the second is recognition of costimulatory or coinhibitory signals. Mesenchymal cells present antigens to T cells but provide a coinhibitory signal, thus specifically inhibiting T cells that recognize them, and other cells expressing similar antigens. Finally, the fact that CD34+ cells express the T cell killing molecule FasL has been postulated as another mechanism of tolerogenesis. Indeed transplantation of bone marrow from mice with a mutated FasL did not induce tolerogenesis in recipients (George, et al., 1998, Nat Med 4:333-335, which is incorporated by reference herein in its entirety).
The potential of using hematopoietic cell transplantation for autoimmunity derives from the belief that the immune system can be deleted and recapitulated, but in such a manner to “reset the clock” so that autoreactive T cells will not re-appear (Muraro, et al, Renewing the T cell repertoire to arrest autoimmune aggression. Trends Immunol., e-published on Jan. 4, 2006, which is incorporated by reference herein in its entirety). Specifically, it is known that the process of autoimmunity requires the failure of several self-tolerance mechanisms before clinical presentation appears. These include: a) self-reactive T cell deletion in the thymus; b) anergy/deletion of self reactive T cells in the periphery; c) failure of the regulatory T cell activity; and d) the presence of inflammation or antigen release in order to allow expansion of the autoreactive T cell clone. During autoimmunity the failure of all of these systems is usually a culmination of environmental and genetic factors occurring over a protracted period of time. Accordingly if the immune system could be made to “start anew” the normal tolerogenic processes would again be reactivated and the disease would be cured, at least temporarily. To date clinical use of autologous stem cells has been performed for a variety of autoimmune indications, including rheumatoid arthritis (Jantunen, et al., 1999, Scand J Rheumatol 28:69-74, which is incorporated by reference herein in its entirety), multiple sclerosis (Karussis, et al., 2004, J Neurol Sci 223:59-64; Brodsky, et al., 1999, Curr Opin Oncol 11:83-86, each of which is incorporated by reference herein in its entirety), systemic lupus erythromatosis (Brunner, et al., 2002, Arthritis Rheum 46:1580-1584; Burt, et al., 2006, Jama 295:527-535, each of which is incorporated by reference herein in its entirety), and systemic sclerosis (Viganego, et al., 2000, Curr Rheumatol Rep 2:492-500, which is incorporated by reference herein in its entirety). According to a report in 2005, approximately 700 patients in total have received an autologous stem cells for autoimmune diseases with a positive benefit/risk ratio that has led to initiation of phase III prospective randomized controlled trials (Tyndall, et al., 2005, Clin Exp Immunol 141:1-9, which is incorporated by reference herein in its entirety).
Induction of tolerance through hematopoietic stem cell transplantation, either from bone marrow or peripheral blood sources possesses the intrinsic danger of bone marrow failure during ablation of the recipient immune system. Although non-myeloablative protocols are under development, even these carry the risk of immune suppression due to the lymphoablation. Accordingly there is a need in the art to develop novel methods of either expanding hematopoietic stem cells ex vivo in large enough quantities to guarantee graft take, as well as methods of in vivo expanding the stem cells and their progeny so that the period under which the transplant recipient is immunosuppressed is minimized.
Stem cell therapy has also been performed in the context of administration of mesenchymal stem cells, without the hematopoietic component, for induction of tolerance. It was demonstrated in a murine model that flk-1+Sca-1-mesenchymal cell transplantation leads to permanent donor-specific immunotolerance in allogeneic host and results in long-term allogeneic skin graft acceptance (Deng, et al., 2004, Exp Hematol 32:861-867, which is incorporated by reference herein in its entirety). Other studies have shown that mesenchymal stem cells are inherently immunosuppressive through production of PGE-2, interleukin-10 and expression of the tryptophan catabolizing enzyme indoleamine 2,3,-dioxygenase as well as Galectin-1 (Kadri, et al., 2005, Stem Cells Dev 14:204-212; Ryan, et al., 2005, J Inflamm (Lond) 2:8, each of which is incorporated by reference herein in its entirety). These stem cells also have the ability to non-specifically modulate the immune response through the suppression of dendritic cell maturation and antigen presenting abilities (Beyth, et al., 2005, Blood 105:2214-2219; Aggarwal, et al., 2005, Blood 105:1815-1822, each of which is incorporated by reference herein in its entirety). Functional induction of allogeneic T cell apoptosis was also demonstrated using freshly isolated, irradiated, or long-term cultured mesenchymal stem cells (Plumas, et al., 2005, Leukemia 19:1597-1604, which is incorporated by reference herein in its entirety). Others have also demonstrated that mesenchymal stem cells have the ability to preferentially induce expansion of antigen specific T regulatory cells with the CD4+CD25+ phenotype (Maccario, et al., 2005, Haematologica 90:516-525, which is incorporated by reference herein in its entirety). Supporting the potential clinical utility of such cells, it was previously demonstrated that administration of mesenchymal stem cells inhibits antigen specific T cell responses in the murine model of multiple sclerosis, experimental autoimmune encephalomyelitis, leading to prevention and/or regression of pathology (Zappia, et al., 2005, Blood 106:1755-1761, which is incorporated by reference herein in its entirety). Safety of infusing mesenchymal stem cells was illustrated in studies administering 1-2.2×106 cells/kg in order to enhance engraftment of autologous bone marrow cell. No adverse events were associated with infusion, although level of engraftment remained to be analyzed in randomized trials (Koc, et al., 2000, J Clin Oncol 18:307-316, which is incorporated by reference herein in its entirety). In a matched pair analysis study, it was demonstrated that in vitro expanded mesenchymal stem cells reduced both acute and chronic graft versus host disease in the allogeneic bone marrow transplant setting. Clinical administration of mesenchymal stem cells was reported in a patient suffering severe, grade IV graft versus host disease in the liver and gut subsequent to bone marrow transplant. Administration of 2×106 cells/kg on day 73 after bone marrow transplant lead to a long term remission of graft versus host disease, which was maintained at the time of publication, 1 year subsequent to administration of the mesenchymal stem cells (Le Blanc, et al., 2004, Lancet 363:1439-1441, which is incorporated by reference herein in its entirety). A feasibility study in 46 patients receiving mesenchymal cells prior to transplant revealed a favorable safety profile and is encouraging further dose finding studies (Lazarus, et al., 2005, Biol Blood Marrow Transplant 11:389-398, which is incorporated by reference herein in its entirety). Unfortunately, mesenchymal cell expansion is relatively slow and in many situations is not practical for widespread clinical use. The development of novel methods of expanding stem cell populations, as for example the methods thought in the present invention, are likely to increase use of this therapeutically promising cell population.
There is evidence that embryonic stem cells are also capable of inducing immunological tolerance. Indeed, coculture of alloreactive T cells with embryonic T cells demonstrated an antigen-specific inhibitory effect (Li, et al., 2004, Stem Cells 22:448-456, which is incorporated by reference herein in its entirety). Data is still preliminary in this area, and the problem of embryonic stem cells inducing teratomas currently precludes their use for this indication. An alternative method of immune modulation using embryonic stem cells is the generation of defined immunological cells that can be used directly, or tailored to possess specific desired properties through modification of culture conditions or gene manipulation. For example, it was demonstrated that the murine model of multiple sclerosis, experimental autoimmune encephalomyelitis can be successfully treated with dendritic cells generated from embryonic stem cell cultures that have been manipulated to present the MOG autoantigen in the presence of TRAIL, a molecule known to induce T cell apoptosis (Hirata, et al., 2005, J Immunol 174:1888-1897, which is incorporated by reference herein in its entirety). Generation of such tailor-made immunological cells would greatly expand the clinical armamentarium of immunotherapy, however, this is limited by the currently lack of methodologies for expanding stem cells in a GMP/GTP compliant and feasible manner.
One of the main therapeutic uses for stem cells is in the area of regenerative medicine. The concept of regenerative medicine is to restore or enhance the ability of tissues to self-organize and heal themselves following endogenous or exogenous injury. Although examples of the use of stem cells for tissue regeneration are almost limitless, several are overviewed below. This should not be taken as an exhaustive literature review, but rather a general discussion for example purposes in order to stimulate one skilled in the art to further investigate this field.
Bone marrow stem cells have been extensively investigated for repair of myocardial tissue subsequent to infarction. Early studies by Orlic demonstrated that administration of GFP c-kit+, lineage−, bone marrow into ligation induced myocardial infarct area resulted in regeneration of myocardial and endothelial tissue by the donor cells (Orlic, et al., 2001, Nature 410:701-705, which is incorporated by reference herein in its entirety). Subsequent studies have used mesenchymal bone marrow cells treated with the DNA methyltransferase inhibitor 5-aza-cytidine to not only transdifferentiate into myocardial tissue, but also to improve left ventricular ejection fraction and inhibit cardiac remodeling (Tomita, et al., 1999. Circulation 100:II247-256, which is incorporated by reference herein in its entirety). Importantly, similar experiments were performed in porcine models of infarction, also indicating improvement in cardiac function (Tomita, et al., 2002, J Thorac Cardiovasc Surg 123:1132-1140, which is incorporated by reference herein in its entirety). Accordingly, clinical experiments were performed administering autologous bone marrow cells directly into the myocardium during coronary bypass grafting. In a series of experiments initiated in 1999, 5 patients treated had no adverse effects, with objective vascularization enhancement in the area of stem cell administration as detected by nuclear imaging (Hamano, et al., 2001, Jpn Circ J 65:845-847, which is incorporated by reference herein in its entirety). A subsequent study administering AC133 purified bone marrow stem cells into the infarct area in 12 patient during bypass grafting demonstrated a marked improvement in left ventricular ejection fraction, a decreased rate of remodeling, and improved perfusion (Stamm, et al., 2004, Thorac Cardiovasc Surg 52:152-158, which is incorporated by reference herein in its entirety). Administration of stem cells into coronary circulation or directly into the myocardium has also been performed both in the angina setting, as well as subsequent to cardiac infarct in order to enhance angiogenesis, and prevent remodeling, respectively. In patients with end stage angina, administration of autologous bone marrow cells using the NOGA catheter system in 14 patients resulted in improved ejection fraction from a baseline of 20% to 29% (P=0.003) and a reduction in end-systolic volume (P=0.03) in the treated patients. Furthermore, electromechanical mapping revealed significant mechanical improvement of the injected segments (P<0.0005) at 4 months after treatment (Perin, et al., 2003, Circulation 107:2294-2302, which is incorporated by reference herein in its entirety). Improvements were also notably maintained in the same patient population at 1-year follow-up (Perin, E., 2004, Int J Cardiol 95 Suppl 1:S45-46, which is incorporated by reference herein in its entirety). Transcoronary administration of bone marrow cells in patients post-myocardial infarction induced an improvement at 6 months in regional and global LV function, increased thickness of the infarcted wall, and showed a reduction in myocardial remodeling as determined by a decrease in the end-systolic volume (Fernandez-Aviles, et al., 2004, Circ Res 95:742-748, which is incorporated by reference herein in its entirety). In another study, patients post myocardial infarction were transplanted with autologous bone marrow cells via a balloon catheter placed into the infarct-related artery during balloon dilatation (percutaneous transluminal coronary angioplasty), resulting in decreased infarct size, improved wall motion score, and a decrease in ventricular remodeling (Strauer, et al., 2002, Circulation 106:1913-1918, which is incorporated by reference herein in its entirety). Randomized trials are currently underway using autologous bone marrow stem cells for increasing cardiac function post myocardial infarction although results are still controversial and inconclusive (Assmus, et al., 2002, Circulation 106:3009-3017; Cleland, et al., 2006, Eur J Heart Fail 8:105-110, each of which is incorporated by reference herein in its entirety). In addition to bone marrow hematopoietic cells, other types of stem cells have been utilized for improvement in myocardial activity, perfusion, and decreasing ventricular remodeling. These include mesenchymal stem cells (Chen, et al., 2004, Chin Med J (Engl) 117:1443-1448, which is incorporated by reference herein in its entirety), endothelial stem cells (Aoki, et al., 2005, J Am Coll Cardiol 45:1574-1579, which is incorporated by reference herein in its entirety), and skeletal myoblasts (Ye, et al., 2006. Exp Biol Med (Maywood) 231:8-19, which is incorporated by reference herein in its entirety). A limiting factor in presently used cellular therapies for myocardial dysfunction is the lack of ability to induce transdifferentiation of the stem cells into the desired cardiac tissue in a directed manner. Additionally, methods do not exist for expanding sufficient numbers of semi-differentiated progenitor stem cells that possess a high proclivity for repairing the heart. This drawback is in part due to lack of proper culture mediums for expansion of such unique cell populations. The current invention addresses this issue.
The importance of stem cells inducing regeneration of other organ systems has been shown in a variety of settings. In a pathological setting, it was reported that bone marrow derived stem cells are the precursors of stomach epithelial tissue in Helicobacter pylori infected mice that progresses to the develop stomach cancer (Houghton, et al., 2004, Science 306:1568-1571, which is incorporated by reference herein in its entirety). In a therapeutic setting, administration of Green Fluorescent Protein (GFP) bone marrow stem cells into rats with ethanol-induced ulcers resulted in generation of GFP expressing, cytokeratin-positive epithelial cells and vimentin-positive interstitial cells, contributing to a decreased pathology in the stem cell recipients (Komori, et al., 2005, J Gastroenterol 40:591-599, which is incorporated by reference herein in its entirety). The human bone marrow derived Flk1(+)/CD31(−)/CD34(−) cell population was reported to transdifferentiated into a variety of tissues, including stomach epithelium when injected into non-obese diabetic, severe combined immunodeficient (NOD-SCID) mice, thus suggesting human stem cells also possess such transdifferentiation ability (Fang, et al., 2003, J Hematother Stem Cell Res 12:603-613, which is incorporated by reference herein in its entirety). Stomach-homing capacity to injured tissue of human stem cells was demonstrated human mesenchymal stem cells infused systemically in NOD-SCID mice that received radiation to the abdominal area. This resulted in a specific rise in stem cell engraftment exclusively to the irradiated areas (Francois, et al., Local irradiation induces not only homing of human Mesenchymal Stem Cells (hMSC) at exposed sites but promotes their widespread engraftment to multiple organs: A study of their quantitative distribution following irradiation damages. Stem Cells, e-published on Dec. 8, 2005, which is incorporated by reference herein in its entirety) It is anticipated that since stem cells can selectively home to the injured stomach area, addition of factors to allow expansion once already homed into the injured tissue will increase therapeutic efficacy of stem cell therapies. The invention teaches methods of expanding cells that have already homed to an injured tissue.
The use of stem cells has also been applied to liver disease. It is known that partial hepatectomy leads to mobilization of an AC133+ stem cell population in clinical situations (Gehling, et al., 2005, J Hepatol 43:845-853, which is incorporated by reference herein in its entirety). Furthermore, studies using carbon tetrachloride induced liver injury have demonstrated a therapeutic effect of bone marrow flk-1+ cell infusion (Fang, et al., 2004, Transplantation 78:83-88, which is incorporated by reference herein in its entirety). It is believed that liver damage induces expression of several chemokines, including stromal derived factor-1 (SDF-1) which attracts stem cells into the damaged areas (Hatch, et al., 2002, Cloning Stem Cells 4:339-351, which is incorporated by reference herein in its entirety). Therapeutic mobilization of endogenous stem cells using granulocyte colony stimulating factor (G-CSF) has also demonstrated protective effects in liver injury models (Quintana-Bustamante, et al., 2006, Hepatology 43:108-116, which is incorporated by reference herein in its entirety). It is anticipated that since stem cells can selectively home to the injured hepatic area, addition of factors to allow expansion once already homed into the injured tissue will increase therapeutic efficacy of stem cell therapies. The invention teaches methods of expanding cells that have already homed to an injured tissue.
Stem cells have also been useful for treatment of neurological deficiencies in a variety of situations. Administration of fetal stem cells in the form of mesenchphalic tissue into the striatal area of Parkinson's disease (PD) patients have demonstrated that grafted dopaminergic neurons can reinnervate the striatum, restore regulated dopamine release and movement-related frontal cortical activation, and result in observable clinical benefit (Lindvall, et al., 2004, NeuroRx 1:382-393, which is incorporated by reference herein in its entirety). Patients suffering from stroke have also been treated by implantation of autologous mesenchymal stem cells into the middle cerebral arterial territory. Improvements were seen in some functional indexes such as the Barth's score (Bang, et al., 2005, Ann Neurol 57:874-882; Rabinovich, et al., 2005, Bull Exp Biol Med 139:126-128, each of which is incorporated by reference herein in its entirety). A wide variety of neurological indications are currently under investigation for amenability to stem cell therapy (Kulbatski, et al., 2005, Curr Drug Targets 6:111-126; Zhu, et al., 2005, Curr Drug Targets 6:97-110, each of which is incorporated by reference herein in its entirety). Unfortunately, ethical issues associated with the use of fetal tissue, as well as inability to define the activities and functions of neurally injected stem cells hampers progress in the field. Development of novel culture and expansion methodologies for stem cell applications is therefore an important area of issue.